Thermostable BrCas12b Enzymes for One-Pot Nucleic Acid Detection

The patent applies parameter changes by modifying the physical-chemical properties of the Cas enzyme through genetic engineering. Specifically, amino acid substitutions are introduced to alter the enzyme's thermal stability parameters, enabling it to maintain structural integrity and catalytic activity at elevated temperatures (60-70°C) required for isothermal amplification, thus resolving the contradiction between detection speed and enzyme stability

The patent creates a composite enzymatic system by combining the engineered Cas enzyme with isothermal amplification components (such as LAMP or RPA reagents) in a single reaction mixture. This composite approach allows the system to function effectively at elevated temperatures where both the amplification reaction and the engineered enzyme remain stable, achieving both rapid detection and enzyme stability

The patent merges the nucleic acid amplification step and the CRISPR-based detection step into a single isothermal reaction mixture. The engineered Cas enzyme maintains stability and activity at the elevated temperature required for isothermal amplification, allowing both amplification and detection to occur simultaneously in one pot, thereby reducing assay complexity and time while maintaining reaction optimization

The patent creates a multi-functional reaction system where a single reaction mixture performs both nucleic acid amplification and target detection functions. The engineered Cas enzyme serves dual purposes: it remains stable during isothermal amplification and subsequently performs specific target recognition and cleavage, eliminating the need for separate optimized reaction conditions and reducing overall assay complexity